Phosphono-carboxylate compounds for treating amyloidosis

ABSTRACT

Therapeutic compounds and methods for modulating amyloid deposition in a subject, whatever its clinical setting, are described. Amyloid deposition is modulated by the administration to a subject of an effective amount of a therapeutic compound comprising a phosphonate group and a carboxylate group, a congener thereof, or a pharmaceutically acceptable salt or ester thereof. In preferred embodiments, an interaction between an amyloidogenic protein and a basement membrane constituent is modulated.

RELATED APPLICATIONS

This application is a continuation application of application Ser. No.10/226,613 filed on Aug. 23, 2002, which in turn is a continuationapplication of application Ser. No. 09/860,255 filed on May 17, 2001,Granted, which in turn is a divisional application of application Ser.No. 09/288,583 filed on Apr. 8, 1999, Granted, which claims benefit ofprovisional application No. 60/081,402 filed on Apr. 10, 1998. Thecontents of all of the aforementioned application(s) are herebyincorporated by reference.

BACKGROUND OF INVENTION

Amyloidosis refers to a pathological condition characterized by thepresence of amyloid. Amyloid is a generic term referring to a group ofdiverse but specific extracellular protein deposits which are seen in anumber of different diseases. Though diverse in their occurrence, allamyloid deposits have common morphologic properties, stain with specificdyes (e.g., Congo red), and have a characteristic red-green birefringentappearance in polarized light after staining. They also share commonultrastructural features and common x-ray diffraction and infraredspectra.

Amyloidosis can be classified clinically as primary, secondary, familialand/or isolated. Primary amyloidosis appears de novo without anypreceding disorder. Secondary amyloidosis is that form which appears asa complication of a previously existing disorder. Familial amyloidosisis a genetically inherited form found in particular geographicpopulations. Isolated forms of amyloidosis are those that tend toinvolve a single organ-system. Different amyloids are also characterizedby the type of protein present in the deposit. For example,neurodegenerative diseases such as scrapie, bovine spongiformencephalitis, Creutzfeldt-Jakob disease and the like are characterizedby the appearance and accumulation of a protease-resistant form of aprion protein (referred to as AScr or PrP-27) in the central nervoussystem. Similarly, Alzheimer's disease, another neurodegenerativedisorder, is characterized by congophilic angiopathy, neuritic plaquesand neurofibrillary tangles, all of which have the characteristics ofamyloids. In this case, the plaque and blood vessel amyloid is formed bythe beta protein. Other systemic or localized diseases such asadult-onset diabetes, complications of long-term hemodialysis andsequelae of long-standing inflammation or plasma cell dyscrasias arecharacterized by the accumulation of amyloids systemically. In each ofthese cases, a different amyloidogenic protein is involved in amyloiddeposition.

SUMMARY OF THE INVENTION

This invention provides methods and compositions which are useful in thetreatment of amyloidosis. The methods of the invention involveadministering to a subject a therapeutic compound which inhibits amyloiddeposition. Accordingly, the compositions and methods of the inventionare useful for inhibiting amyloidosis in disorders in which amyloiddeposition occurs. The methods of the invention can be usedtherapeutically to treat amyloidosis or can be used prophylactically ina subject susceptible to amyloidosis. Without wishing to be bound bytheory, it is believed that the methods of the invention are based, atleast in part, on inhibiting an interaction between an amyloidogenicprotein and a constituent of basement membrane to inhibit amyloiddeposition. The constituent of basement membrane can be a glycoproteinor proteoglycan, preferably heparan sulfate proteoglycan. In certainembodiments, a therapeutic compound used in the method of the inventionpreferably can interfere with binding of a basement membrane constituentto a target binding site on an amyloidogenic protein, thereby inhibitingamyloid deposition.

The invention relates to phosphonocarboxylate compounds, i.e., compoundswhich include a phosphonate group and a carboxylate group, or apharmaceutically acceptable salt or ester thereof. In one embodiment,the method of the invention involves administering to a subject aneffective amount of a therapeutic compound having the formula (FormulaI):

in which Z is XR² or R⁴, R¹ and R² are each independently hydrogen, asubstituted or unsubstituted aliphatic group (preferably a branched orstraight-chain aliphatic moiety having from 1 to 24 carbon atoms in thechain; or an unsubstituted or substituted cyclic aliphatic moiety havingfrom 4 to 7 carbon atoms in the aliphatic ring; preferred aliphatic andcyclic aliphatic groups are alkyl groups, more preferably lower alkyl),an aryl group, a heterocyclic group, or a salt-forming cation; R³ ishydrogen, lower alkyl, aryl, or a salt-forming cation; R⁴ is hydrogen,lower alkyl, aryl or amino (including alkylamino, dialkylamino(including cyclic amino moieties), arylamino, diarylamino, andalkylarylamino); X is, independently for each occurrence, O or S; Y¹ andY² are each independently hydrogen, halogen (e.g., F, Cl, Br, or I),alkyl (preferably lower alkyl), amino, hydroxy, alkoxy, or aryloxy; andn is an integer from 0 to 12 (more preferably 0 to 6, more preferably 0or 1); such that amyloid deposition is modulated.

In preferred embodiments, therapeutic compounds of the invention preventor inhibit amyloid deposition in a subject to which the therapeuticcompound is administered. Preferred therapeutic compounds for use in theinvention include compounds in which both R¹ and R² are pharmaceuticallyacceptable salt-forming cations. It will be appreciated that thestoichiometry of an anionic compound to a salt-forming counterion (ifany) will vary depending on the charge of the anionic portion of thecompound (if any) and the charge of the counterion. In a particularlypreferred embodiment, R¹, R² and R³ are each independently a sodium,potassium or calcium cation. In certain embodiments in which at leastone of R¹ and R² is an aliphatic group, the aliphatic group has between1 and 10 carbons atoms in the straight or branched chain, and is morepreferably a lower alkyl group. In other embodiments in which at leastone of R¹ and R² is an aliphatic group, the aliphatic group has between10 and 24 carbons atoms in the straight or branched chain. In certainpreferred embodiments, n is 0 or 1; more preferably, n is 0. In certainpreferred embodiments of the therapeutic compounds, Y¹ and Y² are eachhydrogen.

In certain preferred embodiments, the therapeutic compound of theinvention can be represented by the formula (Formula II):

in which R¹, R², R³, Y¹, Y², X and n are as defined above. In morepreferred embodiments, the therapeutic compound of the invention can berepresented by the formula (Formula III):

in which R¹, R², R³, Y¹, Y², and X are as defined above, R_(a) and R_(b)are each independently hydrogen, alkyl, aryl, or heterocyclyl, or R_(a)and R_(b), taken together with the nitrogen atom to which they areattached, form a cyclic moiety having from 3 to 8 atoms in the ring, andn is an integer from 0 to 6. In certain preferred embodiments, R_(a) andR_(b) are each hydrogen. In certain preferred embodiments, a compound ofthe invention comprises an α-amino acid (or α-amino acid ester), morepreferably a L-α-amino acid or ester.

In another embodiment, the compounds of the invention can be representedby the formula (Formula IV):

in which G represents hydrogen or one or more substituents on the arylring (e.g., alkyl, aryl, halogen, amino, and the like) and L is asubstituted alkyl group (in certain embodiments, preferably a loweralkyl), more preferably a hydroxy-substituted alkyl or an alkylsubstituted with a nucleoside base.

The therapeutic compounds of the invention are administered to a subjectby a route which is effective for modulation of amyloid deposition.Suitable routes of administration include oral, transdermal,subcutaneous, intravenous, intramuscular and intraperitoneal injection.A preferred route of administration is oral administration. Thetherapeutic compounds can be administered with a pharmaceuticallyacceptable vehicle.

The invention also provides methods for treating a disease stateassociated with amyloidosis by administering to a subject an effectiveamount of a therapeutic compound having the formula described supra,such that a disease state associated with amyloidosis is treated.

The invention provides methods for modulating amyloid depositioncharacterized by interaction between an amyloidogenic protein and aconstituent of a basement membrane by administering to the subject aneffective amount of a therapeutic compound having the formula describedsupra, such that modulation of amyloid deposition characterized byinteraction between an amyloidogenic protein and a constituent of abasement membrane occurs.

The invention further provides pharmaceutical compositions for treatingamyloidosis. The pharmaceutical compositions include a therapeuticcompound of the invention in an amount effective to modulate amyloiddeposition and a pharmaceutically acceptable vehicle.

The invention also provides packaged pharmaceutical compositions fortreating amyloidosis. The packaged pharmaceutical compositions include atherapeutic compound of the invention and instructions for using thepharmaceutical composition for treatment of amyloidosis.

DETAILED DESCRIPTION OF INVENTION

This invention pertains to methods and compositions useful for treatingamyloidosis. The methods of the invention involve administering to asubject a therapeutic compound which modulates amyloid deposition.“Modulation of amyloid deposition” is intended to encompass preventionof amyloid formation, inhibition of further amyloid deposition in asubject with ongoing amyloidosis and reduction of amyloid deposits in asubject with ongoing amyloidosis. Modulation of amyloid deposition isdetermined relative to an untreated subject or relative to the treatedsubject prior to treatment. In certain embodiments, amyloid depositioncan be modulated by modulating an interaction between an amyloidogenicprotein and a constituent of basement membrane.

“Basement membrane” refers to an extracellular matrix comprisingglycoproteins and proteoglycans, including laminin, collagen type IV,fibronectin chondroitan sulfate, and/or heparan sulfate proteoglycan(HSPG). In one embodiment, amyloid deposition is modulated byinterfering with an interaction between an amyloidogenic protein and asulfated glycosaminoglycan such as HSPG. Sulfated glycosaminoglycans areknown to be present in all types of amyloids (see Snow, A. D. et al.(1987) Lab. Invest. 56:120-123) and amyloid deposition and HSPGdeposition occur coincidentally in animal models of amyloidosis (seeSnow, A. D. et al. (1987) Lab. Invest. 56:665-675). In preferredembodiments of the methods of the invention, molecules which have asimilar structure to a sulfated glycosaminoglycan are used to modulateinteraction between an amyloidogenic protein and basement membraneconstituent. In particular, the therapeutic compounds of the inventionpreferably comprise at least one phosphonate group (or phosphonicester), or a functional equivalent thereof (includingphosphorus-containing anionic groups including, but not limited to,phosphates, phosphate esters, phosphinates, and the like), and acarboxylate group or carboxylic ester (or a congener such as a thioacid,thiolester, or thionoester), provided that the compound includes, or iscapable of having after reaction in vivo, at least one anionic group.The anionic groups(s) can optionally be covalently bound to a carrier(e.g., an aliphatic group, peptide or peptidomimetic, or the like). Inaddition to functioning as a carrier for the anionic functionality, thecarrier molecule can enable the compound to traverse biologicalmembranes and to be biodistributed without excessive or prematuremetabolism.

In one embodiment, the method of the invention includes administering tothe subject an effective amount of a therapeutic compound which has atleast one phosphonate group or phosphonic ester group. The therapeuticcompound is preferably capable of modulating interaction between anamyloidogenic protein and a glycoprotein or proteoglycan constituent ofa basement membrane to thus modulate amyloid deposition. The therapeuticcompound has the formula (Formula I):

in which Z is XR² or R⁴, R¹ and R² are each independently hydrogen, asubstituted or unsubstituted aliphatic group (preferably a branched orstraight-chain aliphatic moiety having from 1 to 24 carbon atoms in thechain; or an unsubstituted or substituted cyclic aliphatic moiety havingfrom 4 to 7 carbon atoms in the aliphatic ring; preferred aliphatic andcyclic aliphatic groups are alkyl groups, more preferably lower alkyl),an aryl group, a heterocyclic group, or a salt-forming cation; R³ ishydrogen, lower alkyl, aryl, or a salt-forming cation; X is,independently for each occurrence, O or S; R⁴ is hydrogen, lower alkyl,aryl or amino; Y¹ and Y² are each independently hydrogen, halogen (e.g.,F, Cl, Br, or I), lower alkyl, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), hydroxy,alkoxy, or aryloxy; and n is an integer from 0 to 12 (more preferably 0to 6, more preferably 0 or 1); such that amyloid deposition ismodulated.

In preferred embodiments, therapeutic compounds of the invention preventor inhibit amyloid deposition in a subject to which the therapeuticcompound is administered. Preferred therapeutic compounds for use in theinvention include compounds in which both R¹ and R² are pharmaceuticallyacceptable salt-forming cations. It will be appreciated that thestoichiometry of an anionic compound to a salt-forming counterion (ifany) will vary depending on the charge of the anionic portion of thecompound (if any) and the charge of the counterion. In a particularlypreferred embodiment, R¹, R² and R³ are each independently a sodium,potassium or calcium cation. In certain embodiments in which at leastone of R¹ and R² is an aliphatic group, the aliphatic group has between1 and 10 carbons atoms in the straight or branched chain, and is morepreferably a lower alkyl group. In other embodiments in which at leastone of R¹ and R² is an aliphatic group, the aliphatic group has between10 and 24 carbons atoms in the straight or branched chain. In certainpreferred embodiments, n is 0 or 1; more preferably, n is 0. In certainpreferred embodiments of the therapeutic compounds, Y¹ and Y² are eachhydrogen.

In certain preferred embodiments, the therapeutic compound of theinvention can be represented by the formula (Formula II):

in which R¹, R², R³, Y¹, Y², X and n are as defined above. In morepreferred embodiments, the therapeutic compound of the invention can berepresented by the formula (Formula III):

in which R¹, R², R³, Y¹, Y², and X are as defined above, R_(a) and R_(b)are each independently hydrogen, alkyl, aryl, or heterocyclyl, or R_(a)and R_(b), taken together with the nitrogen atom to which they areattached, form a cyclic moiety having from 3 to 8 atoms in the ring, andn is an integer from 0 to 6. In certain preferred embodiments, R_(a) andR_(b) are each hydrogen. In certain preferred embodiments, a compound ofthe invention comprises an α-amino acid (or α-amino acid ester), morepreferably a L-α-amino acid or ester.

The Z, Q, R¹, R², R³, Y¹, Y² and X groups are each independentlyselected such that the biodistribution of the compound for an intendedtarget site is not prevented while maintaining activity of the compound.For example, the number of anionic groups (and the overall charge on thetherapeutic compound) should not be so great as to inhibit traversal ofan anatomical barrier, such as a cell membrane, or entry across aphysiological barrier, such as the blood-brain barrier, in situationswhere such properties are desired. For example, it has been reportedthat esters of phosphonoformate have biodistribution propertiesdifferent from, and in some cases superior to, the biodistributionproperties of phosphonoformate (see, e.g., U.S. Pat. Nos. 4,386,081 and4,591583 to Helgstrand et al., and U.S. Pat. Nos. 5,194,654 and5,463,092 to Hostetler et al.). Thus, in certain embodiments, at leastone of R¹ and R² is an aliphatic group (more preferably an alkyl group),in which the aliphatic group has between 10 and 24 carbons atoms in thestraight or branched chain. The number, length, and degree of branchingof the aliphatic chains can be selected to provide a desiredcharacteristic, e.g., lipophilicity. In other embodiments, at least oneof R¹ and R² is an aliphatic group (more preferably an alkyl group), inwhich the aliphatic group has between 1 and 10 carbons atoms in thestraight or branched chain. Again, the number, length, and degree ofbranching of the aliphatic chains can be selected to provide a desiredcharacteristic, e.g., lipophilicity or ease of ester cleavage byenzymes. In certain embodiments, a preferred aliphatic group is an ethylgroup.

It has also been reported that certain thiophosphate compounds have invivo activity as anti-viral agents which is equal to or greater than theactivity of the corresponding oxy-phosphate compounds (possibly due todifferences in bioavailability of the compounds). Accordingly, incertain preferred embodiments, the therapeutic compound includes amoiety selected from the group consisting of —P(S)(OR¹)(OR²),—P(S)(SR¹)(OR²), or —P(S)(SR¹)(SR²).

In another embodiment, compounds useful in the methods of the inventioncan be represented by the formula (Formula IV):

In another embodiment, the compounds of the invention can be representedby the formula (Formula IV):

in which G represents hydrogen or one or more substituents on the arylring (e.g., alkyl, aryl, halogen, amino, and the like) and L is asubstituted alkyl group (in certain embodiments, preferably a loweralkyl), more preferably a hydroxy-substituted alkyl or an alkylsubstituted with a nucleoside base. In certain embodiments, G ishydrogen or an electron-donating group. In embodiments in which G is anelectron-withdrawing group, G is preferably an electron withdrawinggroup at the meta position. The term “electron-withdrawing group” isknown in the art, and, as used herein, refers to a group which has agreater electron-withdrawing than hydrogen. A variety ofelectron-withdrawing groups are known, and include halogens (e.g.,fluoro, chloro, bromo, and iodo groups), nitro, cyano, and the like.Similarly, the term “electron-donating group”, as used herein, refers toa group which is less electron-withdrawing than hydrogen. In embodimentsin which G is an electron donating group, G can be in the ortho, meta orpara position.

In certain preferred embodiments, L is a moiety selected from the groupconsisting of (Formulas IVa-IVg):

Table 1 lists data pertinent to the characterization of these compoundsusing art-recognized techniques. TABLE 1 COMPOUND ³¹P NMR ¹³C NMRFAB-MS(−) IVa −6.33(DMSO-d₆) 60.97 CH₂OH(d, J=6Hz) 245.2 66.76 CHOH(d,J=7.8Hz) 121.65, 121.78, 121.99, 125.71, 129.48, 129.57, 126.43 AromaticCH 134.38 Aniline C—N 150.39 Phenyl C—O(d, J=7Hz) 171.57 P—C═O(d,J=234Hz) IVb −6.41(DMSO-d₆) 13.94 CH₃ 456 22.11, 24.40, 28.56, 28.72,28.99, 29.00, 31.30, 33.43, —(CH₂)₁₀— 65.03 CH₂—OC(O) 66.60 CH₂—OP(d,J=5.6Hz) 67.71 CH₂—OH(d, J=6Hz) 121.73, 121.10, 125.64, 126.57, 129.40,129.95, Aromatic CH 134.04 Aniline C—N 150.31 Phenyl C—O 171.44 P—C═O(d,J=6.7Hz) 172.83 O—C═O IVc −6.46(DMSO-d₆) 13.94 CH₃ 471 22.11, 25.10,28.68, 28.72, 28.85, 29.00, 30.76, 31.31, 32.10, —(CH₂)₁₀— 43.36 CH₂—S68.43 CH₂—OH 68.43 CH—OH(d, J=6.3Hz) 68.76 P—O—CH₂-9d, J=5.8Hz) 121.75,122.03, 125.62, 126.37, 129.30, 129.53, Aromatic CH 134.23 Aniline C—N150.37 Phenyl C—O(d, J=6.7Hz) 171.47 P—C═O(d, J=234.0Hz) 198.47 S—C═OIVd −6.61(DMSO-d₆) _(13.94) CH₃ 416 22.06, 25.14, 28.24, 28.35, 31.09,32.14 —CH₂)_(6—) 43.40 CH₂—S 68.50 P—O—CH₂—(d, J=5.8Hz) 68.77 CH—OH(d,6.4Hz) 121.78, 122.59, 125.69, 127.06, 129.43, 129.59 Aromatic CH 133.39Aniline C—N 150.38 Phenyl C—O(d, J=6.7Hz) 171.47 P—C═O(d, J=234.4Hz)198.54 S—C═O IVe −5.76(D₂O) N/A N/A IVf −7.00(DMSO-d₆) N/A N/A IVg−6.60(DMSO-D6) 70.84 CH2—OH 321 72.17 CH—OH 121.68, 121.79, 121.85,125.71 127.10, 127.92, 129.36, 129.50, 129.59 Aromatic CH 134.51 AnilineC—N 142.34 Aromatic C—CH 150.37 Phenyl C—O(d, J=6.2Hz) 171.59 P—C═O(d,J=232.6Hz)

In another aspect, the invention includes novel compounds useful forinhibiting amyloidosis, and/or compounds having antiviral activity. Thecompounds of the invention can be represented by the structures ofFormula IV, e.g., a compound of Formula IV in which G is hydrogen (e.g.,the phenyl ring is unsubstituted) and L is any of the moieties ofFormulas IVa-IVg. A more preferred compound is the compound of FormulaIVc.

In another aspect, the invention provides a method for preparing estersof phosphonates, e.g., phosphono-carboxylate compounds of the invention,e.g., a compound of Formula IV in which G is hydrogen and L is a moietyof Formula IVa-IVg. Illustratively, the method includes the step ofreacting a phosphonodichloridate (or other phosphonate diacid halide)with a disilylated diol under conditions such that a compound of ForumlaIV is formed (see Example 2, infra).

Thus, in one embodiment, the invention provides a method for preparing acompound represented by the Formula (Formula V):

in which R is alkyl or aryl, and R′ is hydrogen, alkyl, or aryl(including heteroaromatic groups such as nucleosides). The methodincludes the step of reacting an ester of a carbonylphosphono diacidhalide (e.g., ROOC—P(O)(A)(A′), in which R is as described in Formula V,and A and A′ are both halogen or other good leaving groups, e.g.,chloro, iodo, bromo, pentafluorophenyl, and the like, which can be thesame or different) with a disilylether of a vicinal diol, underconditions such that the compound of Formula V is prepared.

An anionic group (i.e., a phosphonate or carboxylate group) of atherapeutic compound of the invention is a negatively charged moietythat, in certain preferred embodiments, can modulate interaction betweenan amyloidogenic protein and a glycoprotein or proteoglycan constituentof a basement membrane to thus modulate amyloid deposition.

It will be noted that the structure of some of the compounds of thisinvention includes asymmetric carbon atoms. It is to be understoodaccordingly that the isomers (e.g., enantiomers and diastereomers)arising from such asymmetry are included within the scope of thisinvention. Such isomers can be obtained in substantially pure form byclassical separation techniques and by sterically controlled synthesis.For the purposes of this application, unless expressly noted to thecontrary, a compound shall be construed to include both the R or Sstereoisomers at each chiral center.

The ability of a therapeutic compound of the invention to modulateinteraction between an amyloidogenic protein and a glycoprotein orproteoglycan constituent of a basement membrane can be assessed by an invitro binding assay, such as that described in the Exemplification or inU.S. Pat. No. 5,164,295 by Kisilevsky et al. Briefly, a solid supportsuch as a polystyrene microtiter plate is coated with an amyloidogenicprotein (e.g., serum amyloid A protein or β-amyloid precursor protein(β-APP)) and any residual hydrophobic surfaces are blocked. The coatedsolid support is incubated with various concentrations of a constituentof basement membrane, preferably HSPG, either in the presence or absenceof a compound to be tested. The solid support is washed extensively toremove unbound material. The binding of the basement membraneconstituent (e.g., HSPG) to the amyloidogenic protein (e.g., β-APP) isthen measured using an antibody directed against the basement membraneconstituent which is conjugated to a detectable substance (e.g., anenzyme, such as alkaline phosphatase) by detecting the detectablesubstance. A compound which modulates an interaction between anamyloidogenic protein and a glycoprotein or proteoglycan constituent ofa basement membrane will reduce the amount of substance detected (e.g.,will inhibit the amount of enzyme activity detected).

Preferably, a therapeutic compound of the invention interacts with abinding site for a basement membrane glycoprotein or proteoglycan in anamyloidogenic protein and thereby modulates the binding of theamyloidogenic protein to the basement membrane constituent. Basementmembrane glycoproteins and proteoglycans include laminin, collagen typeIV, fibronectin and heparan sulfate proteoglycan (HSPG). In a preferredembodiment, the therapeutic compound inhibits an interaction between anamyloidogenic protein and HSPG.

In certain embodiments, a therapeutic compound of the inventioncomprises a cation (i.e., in certain embodiments, at least one of R¹, R²or R³ is a cation). If the cationic group is hydrogen, H⁺, then thecompound is considered an acid, e.g., phosphonoformic acid. If hydrogenis replaced by a metal ion or its equivalent, the compound is a salt ofthe acid. Pharmaceutically acceptable salts of the therapeutic compoundare within the scope of the invention. For example, at least one of R¹,R² or R³ can be a pharmaceutically acceptable alkali metal (e.g., Li,Na, or K), ammonium cation, alkaline earth cation (e.g., Ca²⁺, Ba²⁺,Mg²⁺), higher valency cation, or polycationic counter ion (e.g., apolyammonium cation). (See, e.g., Berge et al. (1977) “PharmaceuticalSalts”, J. Pharm. Sci. 66:1-19). It will be appreciated that thestoichiometry of an anionic compound to a salt-forming counterion (ifany) will vary depending on the charge of the anionic portion of thecompound (if any) and the charge of the counterion. Preferredpharmaceutically acceptable salts include a sodium, potassium or calciumsalt, but other salts are also contemplated within theirpharmaceutically acceptable range.

The term “pharmaceutically acceptable esters” refers to the relativelynon-toxic, esterified products of the compounds of the presentinvention. These esters can be prepared in situ during the finalisolation and purification of the compounds or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent; either of which are methods known to those skilled inthe art. Carboxylic acids and phosphonic acids can be converted intoesters according to methods well known to one of ordinary skill in theart, e.g., via treatment with an alcohol in the presence of a catalyst.A preferred ester group (e.g., when R³ is lower alkyl) is an ethyl estergroup.

The term “alkyl” refers to the saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. In preferred embodiments, a straight chain orbranched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g.,C₁-C₃₀ for straight chain, C₃-C₃₀ for branched chain), and morepreferably 20 or fewer. Likewise, preferred cycloalkyls have from 4-10carbon atoms in their ring structure, and more preferably have 4-7carbon atoms in the ring structure. The term “lower alkyl” refers toalkyl groups having from 1 to 6 carbons in the chain, and to cycloalkylshaving from 3 to 6 carbons in the ring structure.

Moreover, the term “alkyl” (including “lower alkyl”) as used throughoutthe specification and claims is intended to include both “unsubstitutedalkyls” and “substituted alkyls”, the latter of which refers to alkylmoieties having substituents replacing a hydrogen on one or more carbonsof the hydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It willbe understood by those skilled in the art that the moieties substitutedon the hydrocarbon chain can themselves be substituted, if appropriate.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above. An “aralkyl” moiety is an alkyl substituted with anaryl (e.g., phenylmethyl (benzyl)).

The term “alkoxy”, as used herein, refers to a moiety having thestructure —O-alkyl, in which the alkyl moiety is described above.

The term “aryl” as used herein includes 5- and 6-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, unsubstituted or substituted benzene, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,pyrazine, pyridazine and pyrimidine, and the like. Aryl groups alsoinclude polycyclic fused aromatic groups such as naphthyl, quinolyl,indolyl, and the like. The aromatic ring can be substituted at one ormore ring positions with such substituents, e.g., as described above foralkyl groups. Preferred aryl groups include unsubstituted andsubstituted phenyl groups.

The term “aryloxy”, as used herein, refers to a group having thestructure —O-aryl, in which the aryl moiety is as defined above.

The term “amino,” as used herein, refers to an unsubstituted orsubstituted moiety of the formula —NR_(a)R_(b), in which R_(a) and R_(b)are each independently hydrogen, alkyl, aryl, or heterocyclyl, or R_(a)and R_(b), taken together with the nitrogen atom to which they areattached, form a cyclic moiety having from 3 to 8 atoms in the ring.Thus, the term “amino” is intended to include cyclic amino moieties suchas piperidinyl or pyrrolidinyl groups, unless otherwise stated. An“amino-substituted amino group” refers to an amino group in which atleast one of R_(a) and R_(b), is further substituted with an aminogroup.

In a preferred embodiment of the compounds of Formulas I-III, R¹ or R²can be (for at least one occurrence) a long-chain aliphatic moiety. Theterm “long-chain aliphatic moiety” as used herein, refers to a moietyhaving a straight or branched chain aliphatic moiety (e.g., an alkyl oralkenyl moiety) having from 10 to 24 carbons in the aliphatic chain,e.g., the long-chain aliphatic moiety is an aliphatic chain of a fattyacid (preferably a naturally-occurring fatty acid). Representativelong-chain aliphatic moieties include the aliphatic chains of stearicacid, oleic acid, linolenic acid, and the like.

The therapeutic compound of the invention can be administered in apharmaceutically acceptable vehicle. As used herein “pharmaceuticallyacceptable vehicle” includes any and all solvents, excipients,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like which arecompatible with the activity of the compound/and are physiologicallyacceptable to the subject. An example of a pharmaceutically acceptablevehicle is buffered normal saline (0.15 molar NaCl). The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the therapeutic compound, use thereof in thecompositions suitable for pharmaceutical administration is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

In certain embodiments, the therapeutic compound of the invention can berepresented by the formula:

in which R¹ and R² are each independently hydrogen, an aliphatic group(preferably a branched or straight-chain aliphatic moiety having from 1to 24 carbon atoms, more preferably 10-24 carbon atoms, in the chain; oran unsubstituted or substituted cyclic aliphatic moiety having from 4 to7 carbon atoms in the aliphatic ring), an aryl group, a heterocyclicgroup, or a salt-forming cation; R³ is hydrogen, lower alkyl, aryl, or asalt-forming cation; Y¹ and Y² are each independently hydrogen, halogen(e.g., F, Cl, Br, or I), lower alkyl, hydroxy, alkoxy, or aryloxy; and nis an integer from 0 to 12; such that amyloid deposition is modulated.In one preferred embodiment, therapeutic compounds of the inventionprevent or inhibit amyloid deposition in a subject to which thetherapeutic compound is administered. Preferred therapeutic compoundsfor use in the invention include compounds in which both R¹ and R² arepharmaceutically acceptable salt-forming cations. In a particularlypreferred embodiment, R¹, R² and R³ are each independently a sodium,potassium or calcium cation, and n is 0. In certain preferredembodiments of the therapeutic compounds, Y¹ and Y² are each hydrogen.Particularly preferred therapeutic compounds are salts ofphosphonoformate. Trisodium phosphonoformate (foscarnet sodium orFoscavir®) is commercially available (e.g., from Astra), and itsclinical pharmacology has been investigated (see, e.g., “Physician'sDesk Reference”, 51st Ed., pp. 541-545 (1997)).

A further aspect of the invention includes pharmaceutical compositionsfor treating amyloidosis. The therapeutic compounds in the methods ofthe invention, as described hereinbefore, can be incorporated into apharmaceutical composition in an amount effective to modulateamyloidosis in a pharmaceutically acceptable vehicle.

The invention further contemplates the use of prodrugs which areconverted in vivo to the therapeutic compounds of the invention (see,e.g., R. B. Silverman, 1992, “The Organic Chemistry of Drug Design andDrug Action”, Academic Press, Chp. 8). Such prodrugs can be used toalter the biodistribution (e.g., to allow compounds which would nottypically cross the blood-brain barrier to cross the blood-brainbarrier) or the pharmacokinetics of the therapeutic compound. Forexample, an anionic group, e.g., a phosphonate or carboxylate, can beesterified, e.g., with an ethyl group or a fatty group, to yield aphosphonic or carboxylic ester. When the phosphonic or carboxylic esteris administered to a subject, the ester can be cleaved, enzymatically ornon-enzymatically, to reveal the anionic group. Such an ester can becyclic, e.g., a cyclic phosphonate, or two or more anionic moieties maybe esterified through a linking group. In a preferred embodiment, theprodrug is a phosphonate or carboxylate. An anionic group can beesterified with moieties (e.g., acyloxymethyl esters) which are cleavedto reveal an intermediate compound which subsequently decomposes toyield the active compound. Furthermore, an anionic moiety (e.g., aphosphonate or carboxylate) can be esterified to a group which isactively transported in vivo, or which is selectively taken up by targetorgans. The ester can be selected to allow specific targeting of thetherapeutic moieties to particular organs, as described below forcarrier moieties. In certain embodiments, as described above, compoundsof the invention can have more than one phosphonic or carboxylic estermoiety, e.g., one phosphonic ester and one carboxylic ester, or aphosphonic diester. In such embodiments, the parent compound may includean anioic group and may be active; however, cleavage of any or all esterfunctionalities may result in an active compound. It will be appreciatedthat in a compound having multiple esterified moieties, the ester groupscan be selected to permit selective cleavage of one or more esterfunctionalities, to unveil one or more anionic groups. The relative easeof cleavage of ester groups is well known; for example, a tert-butyloxyester is generally cleaved more slowly than an ethyl ester under certainconditions. Selection of appropriate moieties to provide a desired rateor order of ester cleavage will be routine to the ordinarily-skilledartisan. Thus, the number of anionic functionalities can be controlledto provide for a selective activity of a compound of the inventionaccording to the rate or order of ester cleavage.

Carrier or substituent moieties useful in the present invention may alsoinclude moieties which allow the therapeutic compound to be selectivelydelivered to a target organ or organs. For example, if delivery of atherapeutic compound to the brain is desired, the carrier molecule mayinclude a moiety capable of targeting the therapeutic compound to thebrain, by either active or passive transport (a “targeting moiety”).Illustratively, the carrier molecule may include a redox moiety, asdescribed in, for example, U.S. Pat. Nos. 4,540,564 and 5,389,623, bothto Bodor. These patents disclose drugs linked to dihydropyridinemoieties which can enter the brain, where they are oxidized to a chargedpyridinium species which is trapped in the brain. Thus, drug accumulatesin the brain. Other carrier moieties include compounds, such as aminoacids or thyroxine, which can be passively or actively transported invivo. Such a carrier moiety can be metabolically removed in vivo, or canremain intact as part of an active compound. Structural mimics of aminoacids (and other actively transported moieties), includingpeptidomimetics, are also useful in the invention. As used herein, theterm “peptidomimetic” is intended to include peptide analogs which serveas appropriate substitutes for peptides in interactions with e.g.,receptors and enzymes. The peptidomimetic must possess not onlyaffinity, but also efficacy and substrate function. That is, apeptidomimetic exhibits function(s) of a peptide, without restriction ofstructure. Peptidomimetics, methods for their preparation and use aredescribed in Morgan et al., “Approaches to the discovery of non-peptideligands for peptide receptors and peptidases,” In Annual Reports inMedicinal Chemistry (Virick, F. J., ed.) pp. 243-253, Academic Press,San Diego, Calif. (1989), the contents of which are incorporated hereinby reference. Many targeting moieties are known, and include, forexample, asialoglycoproteins (see, e.g. Wu, U.S. Pat. No. 5,166,320) andother ligands which are transported into cells via receptor-mediatedendocytosis (see below for further examples of targeting moieties whichmay be covalently or non-covalently bound to a carrier molecule).Furthermore, the therapeutic compounds of the invention may bind toamyloidogenic proteins in the circulation and thus be transported to thesite of action.

The targeting and prodrug strategies described above can be combined toproduce a compound that can be transported as a prodrug to a desiredsite of action and then unmasked to reveal an active compound.

In the methods of the invention, amyloid deposition (e.g., deposition ofβ-amyloid) in a subject is modulated by administering a therapeuticcompound of the invention to the subject. The term “subject” is intendedto include living organisms in which amyloidosis can occur. Examples ofsubjects include humans, monkeys, cows, sheep, goats, dogs, cats, mice,rats, and transgenic species thereof. Administration of the compositionsof the present invention to a subject to be treated can be carried outusing known procedures, at dosages and for periods of time effective tomodulate amyloid deposition in the subject. An effective amount of thetherapeutic compound necessary to achieve a therapeutic effect may varyaccording to factors such as the amount of amyloid already deposited atthe clinical site in the subject, the age, sex, and weight of thesubject, and the ability of the therapeutic compound to modulate amyloiddeposition in the subject. Dosage regimens can be adjusted to providethe optimum therapeutic response. For example, several divided doses maybe administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. A non-limitingexample of an effective dose range for a therapeutic compound of theinvention (e.g., phosphonoformic acid, trisodium salt) is between 0.5and 500 mg/kg of body weight/per day. In an aqueous composition,preferred concentrations for the active compound (i.e., the therapeuticcompound that can modulate amyloid deposition) are between 5 and 500 mM,more preferably between 10 and 100 mM, and still more preferably between20 and 50 mM.

The therapeutic compounds of the invention can be effective whenadministered orally. Accordingly, a preferred route of administration isoral administration. Alternatively, the active compound may beadministered by other suitable routes such as subcutaneous, intravenous,intramuscular or intraperitoneal administration, and the like (e.g. byinjection). Depending on the route of administration, the activecompound may be coated in a material to protect the compound from theaction of acids and other natural conditions which may inactivate thecompound.

The compounds of the invention can be formulated to ensure properdistribution in vivo. For example, the blood-brain barrier (BBB)excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds of the invention cross the BBB, they can beformulated, for example, in liposomes. For methods of manufacturingliposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs (“targetingmoieties”), thus providing targeted drug delivery (see, e.g., V. V.Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moietiesinclude folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low etal.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134);gp120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I.J. Fidler (1994) Immunomethods 4:273. In a preferred embodiment, thetherapeutic compounds of the invention are formulated in liposomes; in amore preferred embodiment, the liposomes include a targeting moiety.

Delivery and in vivo distribution can also be affected by alteration ofan anionic group of compounds of the invention. For example, anionicgroups such as phosphonate or carboxylate can be esterified to providecompounds with desirable pharmocokinetic, pharmacodynamic,biodistributive, or other properties. Exemplary compounds includephosphonoformate trisodium salt (Foscarnet, Foscavir), phosphonoacetate,trisodium salt, and pharmaceutically acceptable salts or esters thereof.

To administer the therapeutic compound by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the therapeutic compound may be administered to a subjectin an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., (1984) J. Neuroimmunol.7:27).

The therapeutic compound may also be administered parenterally (e.g.,intramuscularly, intravenously, intraperitoneally, intraspinally, orintracerebrally). Dispersions can be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The vehicle can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In some cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic compound in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filter sterilization. Generally, dispersions are prepared byincorporating the therapeutic compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient (i.e., the therapeutic compound) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The therapeutic compound can be orally administered, for example, withan inert diluent or an assimilable edible carrier. The therapeuticcompound and other ingredients may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet. For oral therapeutic administration, thetherapeutic compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical vehicle. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofamyloid deposition in subjects.

Therapeutic compositions can be administered in time-release or depotform, to obtain sustained release of the therapeutic compounds overtime. The therapeutic compounds of the invention can also beadministered transdermally (e.g., by providing the therapeutic compound,with a suitable carrier, in patch form).

Active compounds are administered at a therapeutically effective dosagesufficient to modulate amyloid deposition (or amyloid load) in asubject. A “therapeutically effective dosage” preferably modulatesamyloid deposition by at least about 20%, more preferably by at leastabout 40%, even more preferably by at least about 60%, and stilt morepreferably by at least about 80% relative to untreated subjects. Theability of a compound to modulate amyloid deposition can be evaluated inmodel systems that may be predictive of efficacy in modulating amyloiddeposition in human diseases, such as animal model systems known in theart (including, e.g., the method described in PCT Publication WO96/28187) or by in vitro methods, e.g., the method of Chakrabartty,described in PCT Publication WO 97/07402, or the assay described inExample 1, infra. Alternatively, the ability of a compound to modulateamyloid deposition can be evaluated by examining the ability of thecompound to modulate an interaction between an amyloidogenic protein anda basement membrane constituent, e.g., using a binding assay such asthat described hereinabove. Furthermore, the amount or distribution ofamyloid deposits in a subject can be non-invasively monitored in vivo,for example, by use of radiolabelled tracers which can associate withamyloid deposits, followed by scintigraphy to image the amyloid deposits(see, e.g., Aprile, C. et al., Eur. J. Nucl. Med. 22:1393 (1995);Hawkins, P. N., Baillieres Clin. Rheumatol. 8:635 (1994); and referencescited therein). Thus, for example, the amyloid load of a subject can beevaluated after a period of treatment according to the methods of theinvention and compared to the amyloid load of the subject prior tobeginning therapy with a therapeutic compound of the invention, todetermine the effect of the therapeutic compound on amyloid depositionin the subject.

It will be appreciated that the ability of a compound of the inventionto modulate amyloid deposition or amyloid load can, in certainembodiments, be evaluated by observation of one or more symptoms orsigns associated with amyloid deposition or amyloid load in vivo. Thus,for example, the ability of a compound to decrease amyloid deposition oramyloid load may be associated with an observable improvement in aclinical manifestation of the underlying amyloid-related disease stateor condition, or a slowing or delay in progression of symptoms of thecondition. Thus, monitoring of clinical manifestations of disease can beuseful in evaluating the amyloid-modulating efficacy of a compound ofthe invention.

The method of the invention is useful for treating amyloidosisassociated with any disease in which amyloid deposition occurs.Clinically, amyloidosis can be primary, secondary, familial or isolated.Amyloids have been categorized by the type of amyloidogenic proteincontained within the amyloid. Non-limiting examples of amyloids whichcan be modulated, as identified by their amyloidogenic protein, are asfollows (with the associated disease in parentheses after theamyloidogenic protein): β-amyloid (Alzheimer's disease, Down's syndrome,hereditary cerebral hemorrhage amyloidosis [Dutch], cerebralangiopathy); amyloid A (reactive [secondary] amyloidosis, familialMediterranean Fever, familial amyloid nephropathy with urticaria anddeafness [Muckle-Wells syndrome]); amyloid κ L-chain or amyloid λL-chain (idiopathic [primary], myeloma or macroglobulinemia-associated);Aβ2M (chronic hemodialysis); ATTR (familial amyloid polyneuropathy[Portuguese, Japanese, Swedish], familial amyloid cardiomyopathy[Danish], isolated cardiac amyloid, systemic senile amyloidosis); AIAPPor amylin (adult onset diabetes, insulinoma); atrial naturetic factor(isolated atrial amyloid); procalcitonin (medullary carcinoma of thethyroid); gelsolin (familial amyloidosis [Finnish]); cystatin C(hereditary cerebral hemorrhage with amyloidosis [Icelandic]); AApoA-I(familial amyloidotic polyneuropathy [Iowa]); AApoA-II (acceleratedsenescence in mice); fibrinogen-associated amyloid; lysozyme-associatedamyloid; and AScr or PrP-27 (Scrapie, Creutzfeldt-Jacob disease,Gerstmann-Straussler-Scheinker syndrome, bovine spongiformencephalitis).

Compounds for use in the methods of the invention are commerciallyavailable and/or can be synthesized by standard techniques known in theart. In general, phosphonic esters can be prepared from thecorresponding phosphonic acid by standard methods. Similarly, carboxylicesters can be prepared from the free carboxylic acid by standardtechniques (for a reference to esterification techniques, see, e.g., R.Larock, “Comprehensive Organic Transformations,” VCH Publishers (1989)).Carboxylic esters can be converted to thionoesters by known reactions,e.g., by treatment with Lawesson's reagent(2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide,which is commercially available, e.g., from Aldrich Chemical Co.,Milwaukee, Wis.). Compounds of the present invention also can beprepared as described below. The following Examples further illustratethe present invention and are not intended to be further limiting inanyway.

EXAMPLE 1

It is known that amyloidogenic peptides or proteins which have formedamyloid deposits or plaques have a significant amount of β-sheetsecondary structure, while the unaggregated peptide or protein generallyhas less β-sheet structure. It is believed that the ability of acandidate therapeutic compound to prevent the formation of β-sheetsecondary structure in vitro may be correlated with the ability of thecompound to inhibit amyloidogenesis in vivo. Accordingly, phosphonatecompounds were assayed for ability to prevent the formation of β-sheetsecondary structure in assay systems including an in vitro circulardichroism (CD) assay.

Aβ is a 40 amino acid protein associated with Alzheimer's disease. Aβpeptide was prepared and purified as described in Fraser, P. E. et al.,Biochemistry 31, 10716 (1992). Briefly, the peptide was synthesized bystandard solid-phase techniques and purified by HPLC according to wellknown procedures.

All CD experiments were performed on a commercially availableinstrument. The cell was maintained at 25° C. using a circulating waterbath. Computer-averaging of traces was performed to improvesignal-to-noise ratios. The solvent signal was subtracted. CDexperiments were performed for each test compound according to thefollowing procedure:

A stock solution of purified peptide was made by dissolving the peptidein phosphate-buffered saline (PBS) to a concentration of 2 mg/ml. A testsolution was made for each potential therapeutic agent (test compound)as shown below: Aβ stock solution 25 μl Test compound (20 mg/ml) 2.5 μlDistilled water 2.5 μl 10 mM Tris-HCl buffer, pH 7 370 μlThe control sample had no test compound, and a total of 5 μl distilledwater was added. The test solution was incubated for either 0 or 24hours at 37° C. before CD measurement. The size minimum in the CDspectrum at 218 nm is believed to be diagnostic of the presence ofβ-pleated sheet. Comparison of the minimum at 218 nm of a candidatecompound, compared to the minimum of a control sample, is believed to beindicative of the ability of the candidate compound to inhibit theformation of β-pleated sheet.

Using this assay, several candidate compounds were tested.Phosphonoformate sodium salt (foscarnet sodium) was found tosignificantly and reproducibly reduce the amount of β-sheet formation,as measured by the CD assay. Phosphonoacetate was also found to beactive in this assay. Thus, phosphonoformate and phosphonoactetate arebelieved to be inhibitors of amyloid deposition.2-carboxyethylphosphonic acid had a lower ability to prevent β-pleatedsheet formation in this model system.

In a preliminary result in a different assay system (in which thecandidate compound and amyloid peptide were incubated togetherovernight, followed by centrifugation and determination of the amount ofsoluble peptide), phosphonoformate trisodium salt was found to havelittle effect on amyloid peptide solubility; it is believed that thebuffer composition may have interfered with the ability of the compoundto inhibit amyloid deposition.

The neurotoxicity of phosphonoformate trisodium salt was investigated incortical/hippocampal neuronal cultures; no significant toxicity wasnoted at concentrations ranging from 10⁻⁷ M to 10⁻⁴ M.

EXAMPLE 2

The procedure described below is further described in Gorin et al., Tet.Lett. 1997, 38:2791-2794, incorporated herein by reference. Theprocedure has the advantage that the reactivity of the nucleophile(e.g., the hydroxyl groups of a diol which react with the phosphonicacid chloride) is attenuated by use of a silyl ether (e.g., atrimethylsilyl ether), which can improve selectivity.

To a solution of (phenoxycarbonyl)phosphoonodichloridate (5 mmol) in 10ml dry THF cooled in an ice water bath under argon was added a vicinalbis-trimethylsilyl ether (5 mmol) (prepared from the vic-diol, e.g., bytreatment with trimethylsilylchloride (TMSCI) or trimethylsilyltriflate(TMSOTf), available from Aldrich Chemical Co., Milwaukee, Wis.) in 10 mLdry THF. After addition was complete, the reaction mixture was stirredfor one hour at room temperature, and the solvent was evaporated underreduced pressure. The residue was taken up in dioxane containing 90 mgwater (5 mmol), neutralized by adding 5 mmol aniline in 10 mL diozane,and the product precipitated by pouring into 200 mL 1:1 diethylether:hexanes. The solid product was filtered and washed with 1:1diethyl ether:hexanes.

Compounds IVa-IVg were prepared by the above procedure using thecorresponding diols, which are commercially available and/or can bereadily prepared by one of ordinary skill in the art using no more thanroutine experimentation.

EXAMPLE 3

The compounds of Formula IVa, IVc and IVd (in salt forms, e.g.,methylpyridinium salts and/or anilinium salts) were tested in at leastone assay for their ability to inhibit amyloidosis. It was found thatthese compounds showed activity in at least one assay system indicativeof their ability to be an inhibitor of amyloidosis in vivo in both freeor salt forms.

Equivalents

The contents of all references, issued patents, and published patentapplications cited throughout this application are hereby incorporatedby reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

1. A method for modulating amyloid deposition in a subject, comprisingadministering to a subject an effective amount of a therapeutic compoundsuch that modulation of amyloid deposition occurs, wherein thetherapeutic compound has the formula:

in which R¹ and R² are each independently hydrogen, a substituted orunsubstituted aliphatic group, an aryl group, a heterocyclic group, or asalt-forming cation; R³ is hydrogen, lower alkyl, aryl, or asalt-forming cation; R⁴ is hydrogen, lower alkyl, aryl or amino; X is,independently for each occurrence, O or S; Y¹ and Y² are eachindependently hydrogen, halogen, alkyl, amino, hydroxy, alkoxy, oraryloxy; Z is XR² or R⁴; and n is an integer from 0 to 12.